The SDS-PAGE separated proteins were electroblotted on a polyvinylidene difluoride (PVDF) membrane. An anti-His(C-term)-HRP antibody was applied according the manufacturer's instructions (Invitrogen). Chemiluminescence was initiated through the Western Lightning Plus-ECL reagent (Perkin Elmer) and visualized on a ChemiDoc MP station (BioRad).
Chemidoc mp station
Manufactured by Bio-Rad
The ChemiDoc MP station is a versatile imaging system designed for a range of laboratory applications. It features high-resolution digital imaging capabilities, enabling precise capture and analysis of chemiluminescent, fluorescent, and visible light signals from various samples.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software version 5, by Bio-Rad (4 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Bio-Rad (2 mentions)
Luminata immobilon crescendo western hrp substrate, by Merck Group (2 mentions)
Sc 393678, by Abcam (2 mentions)
Anti uqcrc1, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Script reverse transcription supermix, by Bio-Rad (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (2 mentions)
Mastercycler ep realplex, by Eppendorf (2 mentions)
Western lightning plus ecl reagent, by PerkinElmer (1 mentions)
Anti his c term hrp antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using chemidoc mp station
1
Western Blot Protein Detection
2
Protein Extraction and Western Blot Analysis
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Protein extracts were isolated from the hearts of 15-day-olds extracted with RIPA lysis buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM dithiothreitol (DTT), 5 mM EDTA, and Halt Protease and Phosphatase Inhibitor Cocktail from Thermo Fisher Scientific), and the protein concentration was measured using DC Protein Assay kits (Bio-Rad).
Equal quantities of proteins were resolved by SDS-PAGE gels and blotted onto polyvinylidene difluoride membranes (Millipore). Then, the membranes were blocked with 2% bovine serum albumin (BSA) before incubation with the primary and secondary antibodies. The immunoblots were visualized by using Luminata -Immobilon Crescendo Western HRP Substrate (Millipore) on a ChemiDoc MP station (Bio-Rad). Image analysis was performed using Image Lab software version 5.2 (Bio-Rad). The antibodies used for the western blots were as follows: anti-Srsf3 (Abcam, ab198291) anti-Gapdh (Cell Signaling, 2118), anti-Tim50 (Santa-Cruz, sc-393678), anti-Tom20 (Abcam, ab186735), Total OXPHOS antibodies (Abcam, ab110413), and anti-Uqcrc1 (ThermoScientific, PA5-21394).
Equal quantities of proteins were resolved by SDS-PAGE gels and blotted onto polyvinylidene difluoride membranes (Millipore). Then, the membranes were blocked with 2% bovine serum albumin (BSA) before incubation with the primary and secondary antibodies. The immunoblots were visualized by using Luminata -Immobilon Crescendo Western HRP Substrate (Millipore) on a ChemiDoc MP station (Bio-Rad). Image analysis was performed using Image Lab software version 5.2 (Bio-Rad). The antibodies used for the western blots were as follows: anti-Srsf3 (Abcam, ab198291) anti-Gapdh (Cell Signaling, 2118), anti-Tim50 (Santa-Cruz, sc-393678), anti-Tom20 (Abcam, ab186735), Total OXPHOS antibodies (Abcam, ab110413), and anti-Uqcrc1 (ThermoScientific, PA5-21394).
3
Cardiac Gene Expression Quantification
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Total RNA was extracted from hearts using RNeasy Mini kits (Qiagen) according to the manufacturer's protocol. A total of 1000 ng of total RNA was used to prepare cDNA in a 20 µL reaction with Script Reverse Transcription Supermix (Bio-Rad). qPCR was performed on technical duplicates with cDNA diluted 30x and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) via a Mastercycler ep RealPlex (Eppendorf). qPCR analysis was performed by the 2(-ΔΔCt) method, normalizing to RPL30.
Splicing PCR was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs), and 5 L of the cDNA was diluted 30x. Thirty cycles of PCR were used to produce the optimal band detectable in a 2% agarose gel. A ChemiDoc MP station (Bio-Rad) was used to reveal the ethidium bromide-stained agarose gel, and image analysis was performed using Image Lab software version 5.2 (Bio-Rad).
Primers are listed in Appendix Table S3.
Splicing PCR was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs), and 5 L of the cDNA was diluted 30x. Thirty cycles of PCR were used to produce the optimal band detectable in a 2% agarose gel. A ChemiDoc MP station (Bio-Rad) was used to reveal the ethidium bromide-stained agarose gel, and image analysis was performed using Image Lab software version 5.2 (Bio-Rad).
Primers are listed in Appendix Table S3.
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Protein extracts were isolated from the hearts of 15-day-olds extracted with RIPA lysis buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM dithiothreitol (DTT), 5 mM EDTA, and Halt Protease and Phosphatase Inhibitor Cocktail from Thermo Fisher Scientific), and the protein concentration was measured using DC Protein Assay kits (Bio-Rad).
Equal quantities of proteins were resolved by SDS-PAGE gels and blotted onto polyvinylidene difluoride membranes (Millipore). Then, the membranes were blocked with 2% bovine serum albumin (BSA) before incubation with the primary and secondary antibodies. The immunoblots were visualized by using Luminata -Immobilon Crescendo Western HRP Substrate (Millipore) on a ChemiDoc MP station (Bio-Rad). Image analysis was performed using Image Lab software version 5.2 (Bio-Rad). The antibodies used for the western blots were as follows: anti-Srsf3 (Abcam, ab198291) anti-Gapdh (Cell Signaling, 2118), anti-Tim50 (Santa-Cruz, sc-393678), anti-Tom20 (Abcam, ab186735), Total OXPHOS antibodies (Abcam, ab110413), and anti-Uqcrc1 (ThermoScientific, PA5-21394).
Equal quantities of proteins were resolved by SDS-PAGE gels and blotted onto polyvinylidene difluoride membranes (Millipore). Then, the membranes were blocked with 2% bovine serum albumin (BSA) before incubation with the primary and secondary antibodies. The immunoblots were visualized by using Luminata -Immobilon Crescendo Western HRP Substrate (Millipore) on a ChemiDoc MP station (Bio-Rad). Image analysis was performed using Image Lab software version 5.2 (Bio-Rad). The antibodies used for the western blots were as follows: anti-Srsf3 (Abcam, ab198291) anti-Gapdh (Cell Signaling, 2118), anti-Tim50 (Santa-Cruz, sc-393678), anti-Tom20 (Abcam, ab186735), Total OXPHOS antibodies (Abcam, ab110413), and anti-Uqcrc1 (ThermoScientific, PA5-21394).
5
Cardiac Gene Expression Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from hearts using RNeasy Mini kits (Qiagen) according to the manufacturer's protocol. A total of 1000 ng of total RNA was used to prepare cDNA in a 20 µL reaction with Script Reverse Transcription Supermix (Bio-Rad). qPCR was performed on technical duplicates with cDNA diluted 30x and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) via a Mastercycler ep RealPlex (Eppendorf). qPCR analysis was performed by the 2(-ΔΔCt) method, normalizing to RPL30.
Splicing PCR was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs), and 5 L of the cDNA was diluted 30x. Thirty cycles of PCR were used to produce the optimal band detectable in a 2% agarose gel. A ChemiDoc MP station (Bio-Rad) was used to reveal the ethidium bromide-stained agarose gel, and image analysis was performed using Image Lab software version 5.2 (Bio-Rad).
Primers are listed in Appendix Table S3.
Splicing PCR was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs), and 5 L of the cDNA was diluted 30x. Thirty cycles of PCR were used to produce the optimal band detectable in a 2% agarose gel. A ChemiDoc MP station (Bio-Rad) was used to reveal the ethidium bromide-stained agarose gel, and image analysis was performed using Image Lab software version 5.2 (Bio-Rad).
Primers are listed in Appendix Table S3.
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